Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds antigen or epitope whose amino acid sequence is...
Reexamination Certificate
2000-01-25
2001-10-30
Park, Hankyel T. (Department: 1648)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds antigen or epitope whose amino acid sequence is...
C530S350000, C435S007100, C435S007210, C435S007800
Reexamination Certificate
active
06309638
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of this invention is vertebrate netrin proteins and genes which are involved in neural axon outgrowth.
2. Background
In the developing nervous system, axons project considerable distances along stereotyped pathways to reach their targets. Axon growth and guidance depends partly on the recognition of cell-surface and extracellular matrix cues along these pathways. The identification of such nerve cell growth and guidance cues is the holy grail of neurobiology. These are the compounds that tell neurons when to grow, where to grow, and when to stop growing. The medical applications of such compounds are enormous and include modulating neuronal growth regenerative capacity, treating neurodegenerative disease, and mapping (e.g. diagnosing) genetic neurological defects.
Over decades of concentrated research, various hypotheses involving chemo-attractants and repellents, labeled pathways, cell adhesion molecules, etc. have been invoked to explain guidance. Molecules such as N-CAM and N-cadherin have been reported to provide favorable substrates for axon growth and certain sensory axons may be responsive to NGF and NGF-like factors. Recent reports suggest the existence of diffusible chemotropic molecule(s) which influence the pattern and orientation of commissural axon growth.
Relevant Literature
Placzek et al. (1990) Development 110, 19-30; Placzek et al. (1990) Cold Spring Harbor Symposia on Quantitative Biology 55, 279-302.; and Tessier-Lavigne et al. (1988) Nature 336: 775-778 report evidence for diffusible chemotropic molecules which influence the pattern and orientation of commissural axon growth. Gundersen and Barret (1980) JCB 87, 546-554, Lohof et al. (1992) J. Neurosci. 12 (4), 1253-1261 and Zheng et al. (1993) Soc. Neurosci. Abstr 19, 608.9 report neural chemotaxis in response to NGF, cAMP and acetylcholine, respectively. Ishii et al. (1992) Neuron 9, 873-881 disclose a gene, unc-6, derived from C. elegans, which has sequence similarity to the disclosed netrins. Data disclosed in this application was published in Serafini et al (1994) Cell 78, 409-424 and Kennedy et al (1994) Cell 78, 425-435 at page 5, column 1. The work was also reported in
The New York Times
, Section B7, Tuesday, Aug. 16, 1994 and more recently (May 19, 1995) described in Science 268, 971-973 (see also references cited therein).
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to netrins and netrin genes. Netrins are a novel class of proteins which are naturally involved in neural axon guidance. The subject compositions include nucleic acids which encode netrin proteins and hybridization probes and primers capable of hybridizing with netrin genes. Netrins find particular use in modulating neural axon outgrowth. The disclosed compositions also find use variously in screening chemical libraries for regulators of axon outgrowth and orientation, in genetic mapping, as probes for related genes, as diagnostic reagents for genetic neurological disease and in the production of specific cellular and animal systems for the development of neurological disease therapy.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides methods and compositions relating to netrins and netrin genes; including methods and compositions for identifying, purifying, characterizing, and producing netrins and for identifying, characterizing, cloning, expressing, inhibiting the expression of and amplifying netrin genes.
Netrins are characterized by sequence similarity to the disclosed netrins 1 and 2. Using the amino acid sequence search program BLASTP (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410), complete (full length) netrin amino acid sequences provide a Probability P(N) score of less than 1.0e
−200
. In contrast, complete amino acid sequence comparison of a netrin with the evolutionarily related laminin proteins provides P(N) scores exceeding 1.0e
−144
. In addition, netrins generally show at least about 25% overall pair-wise sequence identity with all of the disclosed netrins 1 and 2 and at least about 50% pair-wise sequence identity within domain V. Furthermore, netrins are generally characterized by netrin-specific amino acid sequences invariant across the disclosed netrins 1 and 2 as seen in their amino acid alignments. The subject netrins may be incomplete translates of the disclosed netrin cDNA sequences or deletion mutants of the corresponding conceptual translates, which translates or deletion mutants have the netrin binding activity and specificity described herein.
Netrin peptides of the invention comprise unique portions of the disclosed netrin polypeptides and netrin receptors. A “unique portion” has an amino acid sequence unique to that disclosed in that it is not found in any previously known protein and has a length at least long enough to define a novel peptide. Unique portions are found to vary from about 5 to about 25 residues, preferably from 5 to 10 residues in length, depending on the particular amino acid sequence and are readily identified by comparing the subject portion sequences with known peptide/protein sequence data bases. Preferred unique portions include netrin residues that directly bind and activate (agonize) netrin receptors, especially residues that derive from the EGF-like domains of the disclosed sequences, especially those of the human varieties.
Particular preferred netrin peptides are listed here. These peptides are shown by functional assays disclosed herein to have biological activity including axon outgrowth and/or orienting activity. It is apparent to those of ordinary skill in the art that substitutions of chemically conservative residues can be made while preserving function.
Preferred peptides derived from domain V of netrin 2 and netrin 1:
1. NGH AA/SR (SEQ ID NO:01/02, residues 289-294/265-270)
2. VRD RDD N/SLV (SEQ ID NO:01, residues 296-304)
3. VKD KEQ KLV (SEQ ID NO:02, residues 272-280)
4. KHN TE/AG PE (SEQ ID NO:01/02, residues 308-315/284-291)
5. KPF HYD DRP WQR AT/SA REA NE (SEQ ID NO:01/02, residues 320-338/296-319)
6. NLH ARR (SEQ ID NO:01, residues 345-350)
7. RFN MEL YKL SGR KSG GV (SEQ ID NO:01/02, residues 352-368/328-344)
8. RHN TAG RH (SEQ ID NO:01/02, residues 373-380/349-356)
9. KEG FYR DLS KP/SIS/ TH/DR KA (SEQ ID NO:01/02, residues 385-401/361-377)
10. HPV GAA GK/QT (SEQ ID NO:01/02, residues 408-416/384-392)
11. NQT TGQ (SEQ ID NO:01/02, residues 418-423/394-399)
12. KDG VTG I/LT (SEQ ID NO:01/02, residues 427-434/403-410)
13. AKG Y/FQQ SRS PI/VA P (SEQ ID NO:01/02, residues 439-451/415-427)
Preferred peptides derived from the C terminal domains of netrin 2 and netrin 1:
14. IKI PAI/AN/P (SEQ ID NO:01/02, residues 453-459/429-435)
16. STE A/EPA DCD SYC K (SEQ ID NO:01/02, residues 466-478/442-454)
17. KI/MN MKK YCK/R KDY V/AVQ (SEQ ID NO:01/02, residues 485-499/461-475)
18. KFT I/VNI L/T/ISV YK (SEQ ID NO:01/02, residues 513-523/489-499)
19. CKC PKI/V (SEQ ID NO:01/02, residues 545-550/521-526)
20. ADK S/NSL VIQ WRD (SEQ ID NO:01/02, residues 573-584/549-560)
21. RLR RGD QTL W (SEQ ID NO:01, residues 528-537)
22. RVK RGD NFL W (SEQ ID NO:02, residues 504-513)
Preferred peptides derived from domain VI of netrin 2 and netrin 1:
23. DPC YDE (SEQ ID NO:01/02, residues 40-45/27-30)
24. RCI PE/DF VNA/S AFG KEV (SEQ ID NO:01/02, residues 51-65/38-52)
25. SST CGK PP (SEQ ID NO:01/02, residues 68-75/55-62)
26. A/SSD PKR/K AHP PA/S (SEQ ID NO:01, residues 97-107)
27. LTD LNN PH (SEQ ID NO:01, residues 109-116)
28. LTD LNT AA (SEQ ID NO:02, residues 80-87)
29. NL/MT CWR/Q S—(SEQ ID NO:01/02, residues 117-123/88-94)
The claimed netrins are isolated, partially pure or pure and are typically recombinantly produced. An “isolated” protein for example, is unaccompanied by at least some of the material with which it is associated in its natural state and constitutes at least about 0.5%, preferably at least about 2%, and more preferably at least about 5% by weight of the total protein in a given sample; a partially pure protein constitutes at lea
Dodd Jane
Jessell Thomas
Kennedy Timothy
Placzek Marysia
Serafini Tito
Brown Stacy S.
Osman Richard Aron
Park Hankyel T.
The Regents of the University of California
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